The present invention relates to a class of compounds useful in the diagnosis or radiotherapy of metastatic bone disease, pharmaceutical formulations containing them, their use in the diagnosis of disease and monitoring of disease progression and treatment, and methods for their preparation.
Bone is a common site of metastatic disease with around 70-80% of breast and prostate cancers metastasising to bone. Lung, thyroid, kidney and bladder cancers can also metastasise to bone. Once tumour cells are implanted in the bone marrow they release biochemical mediators which activate osteoblasts. The osteoblastic response detected on a bone scan is a secondary response. Osteoblasts are bone-producing cells implicated in the pathology associated with metastatic bone disease. Activated osteoblasts produce large quantities of collagen that, in addition to its structural role, is important in osteoblast differentiation.
Diagnosis of metastatic bone disease may be achieved by one of four methodsxe2x80x94radiography, CT scanning, radioisotope bone scan or MRI. The radioisotope bone scan has been the standard initial imaging method for the past 25 years. The usual tracer for bone scans is 99mTc-methylene diphosphonate (99mTc-MDP). 99mTc-HMDP (hydroxy-methylenediphosphonate) and 99mTc-HEDP (1-hydroxyethyl-1,1-diphosphonate) may also be used. These agents have broadly similar characteristics. Around 550-750 MBq (15-20 mCi) is injected and high bone uptake (30-50% of the injected dose) occurs within 2 hours. Scans are typically carried out 3-4 hr post administration of agent, due to slow clearance from the blood and/or tissue. Whole body imaging (anterior/posterior) with an acquisition time of 20-30 min produces images of high quality, good resolution and high sensitivity/specificity.
99mTc-MDP is adsorbed onto the calcium of hydroxyapatite in bone. This process is influenced by the levels of osteoblastic activity and by skeletal vascularity. There is preferential uptake at sites of active bone formation, and the amount of accumulation is sensitive to the level of blood flow. The bone scan therefore reflects the metabolic reaction of bone to the disease process, regardless of whether the metabolic activity is neoplastic, traumatic or inflammatory in nature. Thus, the tracer accumulates at any site of elevated bone turnover and the scan is therefore very non-specific.
Osteoblastic metastases resulting in hot spots are detected regardless of size but a cold (photopenic) spot, caused as a result of osteolytic disease, has to reach a certain size to be detected.
The general advantages of the radioisotope scan are a large field of view, low cost, low morbidity, high sensitivity for detection of skeletal metastases, ease of performance on any patient and relatively low total body dose.
Tracer accumulation may occur at any skeletal site with an elevated rate of turnover and in this case does not provide functional or vascular information. As the bone scan has low specificity, the nature of an abnormality cannot be determined from the scan, hence benign and malignant lesions often cannot be distinguished. The technique is also anatomically imprecise. Binding to bone can still occur after tumour cells are dead as collagen is still produced. Consequently there is no distinction between bone healing and tumour progression, with the result that it is difficult to monitor effects of treatment. An increase in the uptake of 99mTc-MDP due to bone healing can be seen up to 6 months after treatment and is known as the flare response.
There is no net production of collagen in osteolytic disease, hence false negatives occurxe2x80x94some or all lesions are missed. Such negative scans need to be re-evaluated with clinical and lab findings. If these are non-conclusive then radiography is used, if this is still non-conclusive then bone biopsy or MRI are used.
The low specificity of 99mTc-MDP means the nature of the abnormality e.g. benign vs malignant lesion cannot be detected. In a patient with known primary tumours, multiple hot spots in the bone scan indicate metastases. However 50% of these hot spots could be other non-metastatic lesions. Therefore a lack of specificity observed with 99mTc-MDP means that positive scans often have to be accompanied by radiographic correlation (a positive radiograph confirms the presence of metastases as the bone scan is more sensitive, but a negative radiograph does not rule them out).
MRI is sometimes chosen, mainly due to its ability to demonstrate abnormalities in bone marrow. However, MRI often cannot distinguish between changes that are due to treatment, fracture and tumour and is less well suited to scanning long bones.
Despite the problems associated with the current radioisotope bone imaging agents, their unique features make them the first choice for screening for metastases in a symptomatic patients. However, a negative scan should always be re-evaluated with clinical and laboratory findings due to the possibility of false negatives. Furthermore, the possibility of a non-metastatic cause of an abnormal scan always needs to be considered. Non-conclusive findings generally lead to supplementary examination with radiography. If diagnosis is still unclear, bone biopsy or MRI will be performed.
There is therefore a need for a diagnostic imaging agent which has specificity for metastatic bone lesions (as opposed to other lesion types), and which can give clinically useful information in a single imaging protocol, without the need for additional testing.
Skeletal metastases may respond to chemotherapy or hormone therapy used to treat the primary tumour. They may also respond to radiation or to agents designed to block bone resorption such as the new class of bisphosphonate (BP) drugs. Bisphosphonates have potent inhibitory effects on bone resorption and are the treatment of choice for hypercalcaemia of malignancy. Treatment can lead to a reduction in the number and rate of skeletal complications in multiple myeloma and advanced breast cancer and can delay the onset of progressive disease in bone following palliative chemotherapy in breast cancer and myeloma. BPs also relieve metastatic bone pain in around 50% of patients but this requires intravenous injection as BPs are not potent enough and not tolerated well when taken orally. Response to treatment can be measured by biochemical markers e.g. excretion of collagen cross-links. Radioisotopes are also used in the treatment of bone metastases [Ben-Josef and Porter, Ann Med. 29, 31-35, (1997); Lewington, Phys Med Biol. 41, 2027-2042 (1996)]. 89Sr has been successfully used in pain palliation. Other bone-seeking isotopes include 32P (side effect of myelotoxicity), 153Sm (complexed with EDTMP) and 186Re (complexed with HEDP).
14C and 3H-labelled ascorbic acid derivatives are known. Yamamoto et al [Appl. Radiat. Isot. 43, 633-639 (1992)] have described the preparation of 6-deoxy-6-[18F]fluoro-L-ascorbic acid (18F-DFA), i.e. an ascorbic acid derivative labelled with the positron emitting isotope [18F] via nucleophilic displacement of a cyclic sulfate with fluoride ion. The biodistribution of this compound has been studied in rats and fibrosarcoma-bearing mice. Yamamoto et al [Radioisotopes, 44, 93-98 (1995)] have also studied the biodistribution of 18F-DFA in Wistar normal rats, ODS rats unable to synthesise ascorbic acid, and Wistar male rats implanted with RG-G6 glioma intracerebrally, and [Nucl. Med. Biol., 23, 479-486 (1996)] the in vivo uptake and distribution of 18F-DFA in rat brains following postischemic reperfusion.
The bone uptake reported for 18F-DFA is very low and there is no suggestion that labelled ascorbic acid derivatives could be useful for either bone imaging in general, or metastatic bone disease imaging in particular. In addition, 18F has a half life of 1.8 hours, and is therefore only usable for a few hours (including synthesis and purification time). Hence any clinical use of such PET (positron emission tomography) agents is limited to a very restricted number of medical sites which possess a cyclotron on site.
The invention includes diagnostic agents for the detection and monitoring of metastatic bone disease as well as radiotherapy of such disease. The agents comprise a modified ascorbic acid labelled with a detectable moiety suitable for external imaging (e.g. by scintigraphy or MRI), such as a radionuclide or a paramagnetic metal ion.
Unmodified ascorbic acid has the formula: 
The agents of the present invention act by accumulating in osteoblast cells present at sites of increased bone turnover. These sites include areas of hyperproliferation associated with metastatic bone disease, as well as other bone pathologies. As the ascorbic acid derivatives are only taken up by osteoblasts at active lesions, they are of high diagnostic and prognostic value for osteoblastic lesions and allow rapid monitoring of disease progress. The use of ascorbates may also prevent the occurrence of false negative scans through visualisation of lytic lesions due to associated osteoblastic activity and also allow early diagnosis of small lesions due to the specific uptake mechanism. Uptake into normal bone will occur and will be useful for localising the lesion site, where uptake will be greatly increased.
In a first aspect, the present invention provides a compound of formula: 
where: X1 is OH or SH or NH2 or -L-Z;
X2 and X3 are the same or different and each is H, C1-4 alkyl, C1-4 fluoroalkyl, benzyl, a protecting group or -L-Z;
X4 is H or C1-4 alkyl;
L is a linker comprising a chain of 0-10 atoms;
Z is a group comprising a detectable moiety;
provided that the compound comprises at least one detectable moiety.
X1 is preferably -L-Z. X2 and X3 are preferably H or C1 alkyl, most preferably both X2 and X3 are H. X4 is preferably H or C1 alkyl, most preferably H. The linker L is suitably a chain of 0-10 atoms of formula (A)m 
where: A is xe2x80x94CR2xe2x80x94, xe2x80x94CRxe2x95x90CRxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, xe2x80x94NRCOxe2x80x94, xe2x80x94CONRxe2x80x94, xe2x80x94O(CO)xe2x80x94, xe2x80x94(CO)Oxe2x80x94, xe2x80x94SO2NRxe2x80x94, xe2x80x94NRSO2xe2x80x94, xe2x80x94OCR2xe2x80x94, xe2x80x94SCR2xe2x80x94, xe2x80x94NRCR2xe2x80x94, a C4-8 cycloheteroalkylene group, a C4-8 cycloalkylene group, a C5-12 arylene group or a C3-12 heteroarylene group;
m is an integer of value 0 to 10;
each R group is independently chosen from H, C1-4 alkyl, C1-4 alkenyl, C1-4 alkynyl, C1-4 alkoxyalkyl or C1-4 hydroxyalkyl.
L preferably comprises a 0-4 atom chain.
Preferred compounds have the defined stereochemistry shown: 
with X1xe2x80x94X4, L and Z as defined above.
The xe2x80x9cdetectable moietyxe2x80x9d is a substance suitable for external imaging after human administration and can be a radionuclide, where the radionuclide is a gamma-emitter that emits gamma radiation that can penetrate soft tissue, a beta-emitter or a low energy X-ray emitter. Radionuclides which are positron emitters such as 11C and 18F are outside the scope of the present invention. 3H and 14C are not suitable radioisotopes for either external imaging or radiotherapy, and are hence also outside the scope of the present invention. The detectable moiety can also be: one or more hyperpolarised atom(s) such as the 13C carbon atom of a 13C-enriched compound for MRI imaging; a paramagnetic moiety as a contrast agent for MRI (e.g. certain metal ions such as gadolinium(III), or manganese (II)); a radiopaque moiety such as iopamidol for X-ray contrast imaging (computer assisted tomography) or an ultrasound contrast agent. Preferably, the detectable moiety is either a radionuclide xcex3-emitter such as 123I, 99mTC, 111In, 113mIn or 67Ga or a hyperpolarised material. Most preferred radionuclide xcex3-emitters are 123I and 99mTc, especially 99mTc. It is also envisaged that certain radionuclides will confer useful radiotherapeutic properties on the labelled ascorbic acid. Thus for example 90Y, 89Sr, 186Re, 188Re, 125I, 131I, 32P or 33P labelled ascorbic acids could be used in the treatment of metastatic bone disease. In such applications the therapeutic effect would be due to the local targeted radioactive dose delivered to specific cells, as opposed to any pharmacological effect due to the ascorbic acid. Whichever detectable moiety is chosen, it is strongly preferred that it is bound to the ascorbic acid in such a way that it does not undergo facile metabolism (either in vivo or in vitro), since such metabolism would result in the biodistribution of the detectable moiety no longer reflecting that of the ascorbic acid.
When the detectable moiety is a hyperpolarised 13C atom, this atom may form an integral part of the chemical structure of the ascorbic acid, or can be attached as a supplemental group. Most other detectable moieties must form supplemental structural elements, and can be attached at the 2, 3, 4, 5 or 6-positions of the ascorbic acid derivative. Preferred positions for the detectable moiety are the 2, 3 and 6-positions, with the 6-position being most preferred.
By the term xe2x80x98protecting groupxe2x80x99 is meant those moieties known to those skilled in the art which would prevent metabolic modification of the ascorbic acid moiety. This may include alkyl, alkoxyalkyl, benzyl or acyl groups. The protecting group may also function to prevent any oxidation or other chemical degradation process of the ascorbic acid hydroxyl groups, and for such purposes is chosen to be sufficiently labile so that the protecting group is cleaved during the labelling or radiolabelling process.
When the detectable moiety is a radioactive or paramagnetic metal, the metal ion is always complexed. This metal complex is preferably achieved by attaching a ligand which binds strongly to metals to the ascorbic acid moiety. Such strongly metal-binding ligands include monodentate compounds which bind well to transition metals such as phosphines, isonitriles or hydrazides, and polydentates such as chelating agents. The ligand-ascorbic acid conjugate is complexed with the radioactive or paramagnetic metal ion, and the metal binds selectively to the ligand, giving a metal complex of the ligand linked to the ascorbic acid derivative.
The chelating agents of the present invention comprise 2-10 metal donor atoms covalently linked together by a non-coordinating backbone. Suitable bidentate chelating agents include bisphosphonates and diphosphines. Bisphosphonate complexes of radiometals have the advantage that the radiometal complex is already targeted to the bone in vivo. Preferred chelating agents have 4-8 metal donor atoms and have the metal donor atoms in either an open chain or macrocyclic arrangement or combinations thereof. Most preferred chelating agents have 4-6 metal donor atoms and form 5- or 6-membered chelate rings when coordinated to the metal centre. Such polydentate and/or macrocyclic chelating agents form stable metal complexes which can survive challenge by endogenous competing ligands for the metal in vivo such as transferrin or plasma proteins. The metal complex should also preferably be of low lipophilicity (since high lipophilicity is often related to non-specific uptake), and exhibit low plasma protein binding since plasma bound label again contributes to undesirable high, non-specific background for the imaging agent.
Examples of suitable chelating agents are diaminedioximes (U.S. Pat. No. 4,615,876) or such chelates incorporating amide donors (WO 94/08949); the tetradentate chelates of WO 94/22816; N2S2 diaminedithiols, diamidedithiols or amideaminedithiols; N3S thioltriamides; N2O2 diaminediphenols; N4 chelates such as tetraamines, macrocyclic amines or amide chelates such as cyclam, oxocyclam (which forms a neutral technetium complex) or dioxocyclam; or dithiosemicarbazones. The above described chelates are particularly suitable for technetium, but are useful for other metals also. Other suitable chelates are described in WO 91/01144, which includes chelates which are particularly suitable for indium, yttrium and gadolinium, especially macrocyclic aminocarboxylate and aminophosphonic acid chelates. Chelates which form non-ionic (i.e. neutral) metal complexes of gadolinium are known and described in U.S. Pat. No. 4,885,363. The chelate may also comprise a short sequence of amino acids such as the Cys/aminoacid/Cys tripeptide of WO 92/13572 or the peptide chelates described in EP 0719790 A2.
When the detectable moiety is a radioactive isotope of iodine, the radioiodine atom is preferably attached via a direct covalent bond to an aromatic ring, such as a benzene ring, or to a vinyl group, since it is known that iodine atoms bound to saturated aliphatic systems are prone to in vivo metabolism and hence loss of the detectable moiety.
Bone metastases may be osteoblastic (10%), osteolytic (65%) or mixed (25%) in appearance. It is believed that the compounds of the present invention are only taken up by activated osteoblasts at sites of increased bone turnover, i.e. active lesions. Such sites include areas of hyperproliferation associated with metastatic bone disease, as well as other bone pathologies. The present compounds are therefore expected to be of high diagnostic and prognostic value for osteoblastic lesions, and to allow rapid monitoring of disease and treatment progress. This is in contrast to prior art [99mTc]-MDP, which accumulates at sites of collagen production long after tumour cells are dead, giving no information about cell viability. The compounds of the present invention may also help to prevent the occurrence of false negative scans, via the visualisation of osteolytic lesions due to associated osteoblastic activity and allow early diagnosis of small lesions due to the specific uptake mechanism. The relatively rapid expected clearance time should allow rapid imaging and high patient throughput. The present compounds may also accumulate in fibroblasts and could hence be useful in the diagnostic imaging of sites of wound repair, and may also be useful in the diagnosis of other bone pathologies, for example osteoporosis and arthritis.
A further aspect of the present invention is the disclosure of novel ascorbic acid derivatives. These may be useful as pharmaceuticals for the treatment of tumours known to accumulate ascorbic acid, in particular bone tumours, and may also be attached to therapeutic radioisotopes or cytotoxic drugs.
Novel ascorbic acid derivatives, including those labelled with non-radioactive 127I, have been prepared, and shown to compete with 14C-labelled ascorbic acid for uptake into murine pre-osteoblast (MC3T3-E1) cells. Such derivatives have essentially identical chemical properties to the radioactive counterparts labelled with radioiodine e.g. 123I or 131I. In addition, a few known compounds have also been synthesised and shown to compete with 14C-labelled ascorbic acid for uptake into MC3T3-E1 cells. Furthermore, a 14C-labelled ascorbic acid derivative (compound 17) has been shown to accumulate in primary rat osteoblasts over a 5 hour period and remains stable over this time. Using autoradiography, accumulation has also been demonstrated in mineralised bone nodules produced by rat osteoblasts over a 21-day culture period. In vivo, the amount of 14C-compound 17 accumulating in the epiphysis of the rat 60 minutes after i.v. injection was compared to that accumulating in the diaphysis and was found to be 2.3-fold greater due to increased osteoblast activity at these sites.
The compounds of the present invention may be prepared as follows. When the detectable moiety is radioactive iodine, the substituent linked to ascorbic acid must include a non-radioactive halogen atom (to permit radioiodine exchange), an activated aromatic ring (e.g. a phenol group), an organometallic precursor compound such as a trialkyltin or trialkylsilyl, an organic precursor such as triazenes or other such moiety known to those skilled in the art. Examples of suitable substituents to which radioactive iodine can be attached are given below: 
Both substituents contain groups which permit facile radioiodine substitution onto the aromatic ring. Alternative substituents containing radioactive iodine can be synthesised by direct iodination via radiohalogen exchange, e.g. 
Groups for substitution of radioiodine can be attached to ascorbic acid as follows. A substituent functionalised with a carboxylic acid group can be reacted with the 6-OH of ascorbic acid to give an ester link [J. Carbohyd. Chem. 17(3) 397-404 (1998)]. Alternatively 6-bromo-6-deoxy-L-ascorbic acid [Suskovic, Croat. Chem. Acta, 58, 231 (1985)] can be reacted with an amino or thiol-functionalised substituent to give an amino [Kralj et al, Eur. J. Med. Chem., 31, 23, (1996)] or thioether [Carbohyd. Res., 134, 321, (1984)] link. 6-Amino-6-deoxy-L-ascorbic acid [Suskovic, Croat. Chem. Acta, 62, 537 (1989)] can be reacted with substituents functionalised with a carboxylic acid or an active ester to give an amide link. Persons skilled in the art will recognise that many alternative syntheses of ascorbic acid derivatives suitable for radioiodination are possible based on this disclosure.
An alternative method for synthesising ascorbic acid derivatives suitable for radioiodination involves rearrangement of a L-gulonate (shown below) under acidic conditions [Crawford et at, Adv. Carbohyd. Chem Biochem., 37, 79 (1980)]. 
The 4,6-isopropylidene protecting group is removed and a substituent suitable for radioiodination is linked to the primary hydroxyl of the L-gulonate via an ether or ester link, for example, and the modified L-gulonate rearranged to give the corresponding ascorbic acid derivative. Alternatively the primary hydroxyl can be substituted for a bromo or amino group for reaction with an appropriately functionalised group suitable for radioiodination, prior to rearrangement to the ascorbic acid.
When the detectable moiety is a radioactive or paramagnetic metal ion, a chelating agent is attached to the ascorbate giving a chelate-ascorbic acid conjugate. Such chelate-ascorbic acid conjugates can be prepared using the bifunctional chelate approach. Thus, it is well known to prepare chelating agents which have attached thereto a functional group (xe2x80x9cbifunctional chelatesxe2x80x9d). Functional groups that have been attached to chelating agents include: amine, thiocyanate, maleimide and active ester such as N-hydroxysuccinimide. Such bifunctional chelates can be reacted with suitable functional groups on the ascorbic acid to form the desired conjugate. Examples of chelate-amine conjugates for diaminedioxime ligands are given in WO 95/19187. In the particular case of ascorbic acid, a chelating agent can be attached at the 6-position as follows. Ascorbic acid can be reacted with a chelate-carboxylic acid conjugate to give a chelate-ascorbic acid derivative linked via an ester bond. 6-COOH-6-deoxy-L-ascorbic acid [Stuber et al, Carbohyd. Res., 60, 25 (1978)] can be reacted with a chelate-amine conjugate, or 6-NH26-deoxy-L-ascorbic acid reacted with a chelate-active ester or chelate-carboxylic acid conjugate to give chelate-ascorbic acid derivatives linked via amide bonds. 6-Br6-deoxy-L-ascorbic acid can be reacted with a chelate-amine or chelate-thiol conjugate to give either an amine or thioether link.
An alternative method for synthesising ascorbic acid derivatives involves rearrangement of an L-gulonate derivative as described above. This reaction can be used in the synthesis of chelate-ascorbic acid conjugates. A chelate can be linked to the L-gulonate using one of the methods described above and the resulting chelate-L-gulonate conjugate rearranged to give the corresponding chelate-ascorbic acid derivative. Persons skilled in the art will recognise that many alternative syntheses of chelate-ascorbic acid conjugates are possible based on this disclosure.
When the detectable moiety is a hyperpolarised atom, such as a hyperpolarised 13C atom, the desired hyperpolarised compound can be prepared by polarisation exchange from a hyperpolarised gas (such as 129Xe or 3He) to a suitable 13C-enriched ascorbic acid derivative. Both [1-13C]- and [2-13C]-labelled ascorbic acid derivatives are known, and have been used to examine transport and redox cycling in human erythrocytes [Himmelreich et al. Biochem. 37, 7578 (1998)]. 13C-enriched ascorbic acid derivatives can also be prepared in an analogous manner to the literature synthetic routes for 14C-labelled ascorbic acid derivatives. Thus, Hornig et al [Int. J. Vit. Nutr. Res. 42, 223 (1972) and ibid 42, 511 (1972)] have studied the autoradiographic biodistribution of [1-14C]-L-ascorbic acid in normally fed and vitamin C deficient guinea pigs following intravenous injection. Karr et al [J. Lab. Comp., 6, 155 (1970)] have also prepared [6-14C]-L-ascorbic acid and [5-14C]-L-ascorbic acid from D-glucose-1-14C and D-glucose2-14C, respectively. Williams et al [Carbohyd. Res., 63, 149 (1978)] describe the synthesis of [4-14C]-L-ascorbic acid from D-[3-14C]glucopyranose and [6-14C]-L-ascorbic acid from D-[1-14C]glucopyranose.
Unlabelled ascorbic acid derivatives of the present invention have been tested for their ability to compete for uptake of 14C-ascorbic acid into MC3T3-E1 cells, a murine pre-osteoblast cell line. MC3T3-E1 cells are grown in tissue culture plates and the appropriate assay solution containing a standard concentration of 14C-ascorbic acid plus a competing concentration of ascorbic acid derivative added to each well. The amount of 14C-ascorbic acid taken up by the cells in 60 min is then measured.
Of compounds 11 to 15, only those containing an iodophenyl, bromophenyl or iodovinyl substituent were found to compete for uptake of 14C-ascorbic acid into MC3T3-E1 cells. Results are given in Table 10. Both Compounds 16 and 17 competed for uptake of 14C-ascorbic acid into MC3T3-E1 cells, although competition by compound 16 was very weak. Although both these compounds are known, neither has been reported in the literature to show competition with 14C-ascorbic acid for uptake into pre-osteoblast or osteoblast cells.
The present invention also relates to kits for the preparation of ascorbic acid derivatives labelled with a detectable moiety. The kits are designed to give sterile products suitable for human administration, e.g. via injection into the bloodstream. Possible embodiments are discussed below. When the detectable moiety is 99mTc, the kit would comprise a vial containing either an ascorbic acid derivative suitable for forming a metal complex with 99mTc or a chelate-ascorbic acid conjugate, together with a pharmaceutically acceptable reducing agent such as sodium dithionite, sodium bisulphite, formamidine sulphonic acid, stannous ion, Fe(II) or Cu(I). The reducing agent is preferably a stannous salt such as stannous chloride or stannous tartrate.
Alternatively, the ascorbic acid derivative or chelating agent-ascorbic acid conjugate could be present as the metal complex of a suitable non-radioactive metal, which, upon addition of the radiometal, undergoes transmetallation (i.e. ligand exchange) giving the desired product. The kit is preferably lyophilised and is designed to be reconstituted with sterile 99mTc-pertechnetate (TcO4xe2x88x92) from a 99mTc radioisotope generator to give a solution suitable for human administration without further manipulation.
The agents for the present invention may also be provided in a unit dose form ready for human injection and could for example be supplied in a pre-filled sterile syringe. When the detectable moiety is a radioactive isotope such as 99mTc, the syringe containing the unit dose would also be supplied with a syringe shield (to protect the operator from potential radioactive dose).
The above kits or pre-filled syringes may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g. cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilisers/antioxidants (such as gentisic acid or para-aminobenzoic acid) or bulking agents for lyophilisation (such as sodium chloride or mannitol).
The structure of compound 10 is given in Scheme 1. The structures of compounds 11 to 28 are given in Tables 1 to 4. The preparation of compounds 10-19 and 21-28 is described in Examples 1 to 6. NMR data for the compounds is given in Tables 4 to 9. The biological properties of Compounds 11 to 18 are shown in Example 7 and Table 10. The biological properties of Compound 17 are further discussed in Examples 8 and 9. 
where: [Diaminediphenol]-linkers- are: 
where: [Pn216]-linkers- are: 
where: [Isopropylamine]-linkers- are: 
where: [Hynic]-linker- is: 